PROJECT SUMMARY/ABSTRACT The biliary epithelium is a complex network of interconnected ducts that increase in diameter from small to large bile ducts. It has been proposed that small cholangiocytes contain a population of biliary committed progenitors, showing expression of various biliary progenitor markers, and incorporate into neo-bile ducts at the sites of injury. When large cholangiocytes are damaged, small, Ca2+-dependent cholangiocytes are activated, acquiring phenotypic and functional features of large cholangiocytes and resulting in the repopulation of the injured large bile ducts. Cholestatic liver diseases (CLDs) such as Primary Biliary Cirrhosis (PBC) and Primary Sclerosing Cholangitis (PSC), are chronic diseases that slowly damage the intrahepatic and extrahepatic bile ducts including both small and large bile ducts. During CLDs and other human diseases, extracellular vesicles (EVs) are released by almost all cells (including hepatobiliary progenitors and cholangiocytes) in resting and activated conditions. Their mechanisms of actions are being elucidated and their potential role in health and disease is drawing increasing attention. However, the specific roles of non-coding RNAs (ncRNAs) in cholangiocytes derived EVs during hepatobiliary tissue repair (especially their characteristics during cholestatic liver injury) still need to be addressed. Based on our compelling data, we propose the central hypothesis that ncRNAs in small cholangiocytes derived extracellular vesicles contribute to the recovery of cholestatic liver injury through induction of growth and anti- senescence/anti-inflammation of hepatobiliary tissues and cells. In this application, we propose the systematic evaluation of stemness dependent ncRNAs as markers in small cholangiocytes derived EVs with the therapeutic potentials for cholestatic liver injury. We will address our central hypothesis by focusing on the following specific aims: 1) To define the biliary extracellular vesicle associated ncRNAs signaling involved in anti-inflammation process in hepatobiliary cells. In this study, we will characterize the interactions between miR-125b/let-7 and uc.338/uc.189 family members; define the role of anti-LPS ncRNA signaling in small cholangiocytes derived extracellular vesicles through flow cytometry and real-time PCR analysis. 2) To determine the effects of SMCC-EV associated ncRNAs on accelerating the morphologic and functional recovery of cholestatic liver injury in CLD mice induced in vivo by BDL or in the genetic mouse model of PSC and PBC, Mdr2-/- and dnTGFbRII, respectively. Therapeutic effects of EVs derived from SMCCs with miRNAs or inhibition of T-UCRs on hepatobiliary cell proliferation and inflammation, senescence and fibrosis will be evaluated. The results of the proposed studies may lead to new therapeutic strategies for human cholestatic liver diseases. Meanwhile, the acquired fundamental new knowledge about regulation of growth and tissue repair during cholestatic liver damage by hepatobiliary progenitors derived EVs is expected to advance the general field of anti-inflammatory biology and emerging therapeutic opportunities.